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Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis.

Kumari S, Redouane Y, Lopez-Mosqueda J, Shiraishi R, Romanowska M, Lutzmayer S, Kuiper J, Martinez C, Dikic I, Pasparakis M, Ikeda F - Elife (2014)

Bottom Line: Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation.Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role.Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, Center for Molecular Medicine, University of Cologne, Cologne, Germany.

ABSTRACT
Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.

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Tumor necrosis factor receptor 1 (TNFR1) signaling in keratinocytes triggers chronic proliferative dermatitis phenotype in Sharpincpdm/cpdm mice.(A) Flow cytometric analysis of TNFR1 expression on the isolated keratinocytes from mice with the indicated genotypes. (B and C) Macroscopic pictures, Hematoxylin and Eosin staining (H&E), Keratin 6, 14, 10 and Loricrin as well as cleaved caspase-3 and F4/80 staining of the skin sections from 14-week-old littermate mice of the indicated genotypes. The scale bars are 100 μm. (D) Microscopic quantification of the epidermal thickness from 12–18-week-old mice of the indicated genotypes and their littermate controls (Ctr), which consisted of the following genotypes: Sharpincpdm/wt;Tnfrsf1a−/−, Sharpinwt/wt;Tnfrsf1a−/−, Sharpincpdm/wt;Tnfrsf1afl/fl, Sharpincpdm/wt;TNFR1E-KO, and Sharpinwt/wt;TNFR1E-KO. The Sharpincpdm/cpdm group consisted of Sharpincpdm/cpdm;Tnfrsf1afl/fl and Sharpincpdm/cpdm;Tnfrsf1afl/wt mice that were littermates of the Sharpincpdm/cpdm;TNFR1E-KO mice. The Sharpincpdm/cpdm;Tnfrsf1a−/− mice were derived from a different line and shown here is the picture and the staining from the age-matched mice. Bars represent mean values ± SEM. Statistical significance was determined using the Student's t test (***p ≤ 0.001).DOI:http://dx.doi.org/10.7554/eLife.03422.003
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fig1: Tumor necrosis factor receptor 1 (TNFR1) signaling in keratinocytes triggers chronic proliferative dermatitis phenotype in Sharpincpdm/cpdm mice.(A) Flow cytometric analysis of TNFR1 expression on the isolated keratinocytes from mice with the indicated genotypes. (B and C) Macroscopic pictures, Hematoxylin and Eosin staining (H&E), Keratin 6, 14, 10 and Loricrin as well as cleaved caspase-3 and F4/80 staining of the skin sections from 14-week-old littermate mice of the indicated genotypes. The scale bars are 100 μm. (D) Microscopic quantification of the epidermal thickness from 12–18-week-old mice of the indicated genotypes and their littermate controls (Ctr), which consisted of the following genotypes: Sharpincpdm/wt;Tnfrsf1a−/−, Sharpinwt/wt;Tnfrsf1a−/−, Sharpincpdm/wt;Tnfrsf1afl/fl, Sharpincpdm/wt;TNFR1E-KO, and Sharpinwt/wt;TNFR1E-KO. The Sharpincpdm/cpdm group consisted of Sharpincpdm/cpdm;Tnfrsf1afl/fl and Sharpincpdm/cpdm;Tnfrsf1afl/wt mice that were littermates of the Sharpincpdm/cpdm;TNFR1E-KO mice. The Sharpincpdm/cpdm;Tnfrsf1a−/− mice were derived from a different line and shown here is the picture and the staining from the age-matched mice. Bars represent mean values ± SEM. Statistical significance was determined using the Student's t test (***p ≤ 0.001).DOI:http://dx.doi.org/10.7554/eLife.03422.003

Mentions: Previous studies showed that TNF is required for the development of multi-organ inflammation in Sharpincpdm/cpdm mice (Gerlach et al., 2011). To address whether this function of TNF is mediated by TNFR1, we crossed Sharpincpdm/cpdm mice with Tnfrsf1a−/− animals. Double deficient Sharpincpdm/cpdm;Tnfrsf1a−/− mice did not develop skin inflammation, demonstrating that TNF-induced TNFR1 signaling is essential for the pathogenesis of inflammatory skin lesions in Sharpincpdm/cpdm mice (Figure 1). We then tried to identify the cellular target of the pathogenic TNFR1 signaling in Sharpincpdm/cpdm mice. We have recently shown that TNFR1 signaling in NF-κB-deficient epidermal keratinocytes drives psoriasis-like skin inflammation in mice (Kumari et al., 2013), identifying keratinocytes as an important cellular target of pathogenic TNF signaling in skin inflammation. To address whether TNFR1 signaling in epidermal keratinocytes drives the skin inflammation in Sharpincpdm/cpdm mice, we crossed Sharpincpdm/cpdm mice with K14Cre-Tnfrsf1afl/fl (TNFR1E-KO) mice that lack TNFR1 specifically in keratinocytes (Figure 1A). These Sharpincpdm/cpdm;TNFR1E-KO mice did not develop any macroscopic signs of skin inflammation (Figure 1B). In addition, histological analysis of Sharpincpdm/cpdm;TNFR1E-KO mice skin revealed a normal epidermis without keratinocyte death (cleaved caspase-3 staining in Figure 1C), skin inflammation (F4/80 staining in Figure 1C), or epidermal hyperplasia (H&E, Keratin 6, Keratin 10, and Loricrin staining in Figure 1C and quantification in Figure 1D), similar to Sharpincpdm/cpdm;Tnfrsf1a−/− animals (Figure 1C,D). These results demonstrate that TNFR1 signaling in epidermal keratinocytes is essential for the pathogenesis of skin inflammation in Sharpincpdm/cpdm mice.10.7554/eLife.03422.003Figure 1.Tumor necrosis factor receptor 1 (TNFR1) signaling in keratinocytes triggers chronic proliferative dermatitis phenotype in Sharpincpdm/cpdm mice.


Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis.

Kumari S, Redouane Y, Lopez-Mosqueda J, Shiraishi R, Romanowska M, Lutzmayer S, Kuiper J, Martinez C, Dikic I, Pasparakis M, Ikeda F - Elife (2014)

Tumor necrosis factor receptor 1 (TNFR1) signaling in keratinocytes triggers chronic proliferative dermatitis phenotype in Sharpincpdm/cpdm mice.(A) Flow cytometric analysis of TNFR1 expression on the isolated keratinocytes from mice with the indicated genotypes. (B and C) Macroscopic pictures, Hematoxylin and Eosin staining (H&E), Keratin 6, 14, 10 and Loricrin as well as cleaved caspase-3 and F4/80 staining of the skin sections from 14-week-old littermate mice of the indicated genotypes. The scale bars are 100 μm. (D) Microscopic quantification of the epidermal thickness from 12–18-week-old mice of the indicated genotypes and their littermate controls (Ctr), which consisted of the following genotypes: Sharpincpdm/wt;Tnfrsf1a−/−, Sharpinwt/wt;Tnfrsf1a−/−, Sharpincpdm/wt;Tnfrsf1afl/fl, Sharpincpdm/wt;TNFR1E-KO, and Sharpinwt/wt;TNFR1E-KO. The Sharpincpdm/cpdm group consisted of Sharpincpdm/cpdm;Tnfrsf1afl/fl and Sharpincpdm/cpdm;Tnfrsf1afl/wt mice that were littermates of the Sharpincpdm/cpdm;TNFR1E-KO mice. The Sharpincpdm/cpdm;Tnfrsf1a−/− mice were derived from a different line and shown here is the picture and the staining from the age-matched mice. Bars represent mean values ± SEM. Statistical significance was determined using the Student's t test (***p ≤ 0.001).DOI:http://dx.doi.org/10.7554/eLife.03422.003
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fig1: Tumor necrosis factor receptor 1 (TNFR1) signaling in keratinocytes triggers chronic proliferative dermatitis phenotype in Sharpincpdm/cpdm mice.(A) Flow cytometric analysis of TNFR1 expression on the isolated keratinocytes from mice with the indicated genotypes. (B and C) Macroscopic pictures, Hematoxylin and Eosin staining (H&E), Keratin 6, 14, 10 and Loricrin as well as cleaved caspase-3 and F4/80 staining of the skin sections from 14-week-old littermate mice of the indicated genotypes. The scale bars are 100 μm. (D) Microscopic quantification of the epidermal thickness from 12–18-week-old mice of the indicated genotypes and their littermate controls (Ctr), which consisted of the following genotypes: Sharpincpdm/wt;Tnfrsf1a−/−, Sharpinwt/wt;Tnfrsf1a−/−, Sharpincpdm/wt;Tnfrsf1afl/fl, Sharpincpdm/wt;TNFR1E-KO, and Sharpinwt/wt;TNFR1E-KO. The Sharpincpdm/cpdm group consisted of Sharpincpdm/cpdm;Tnfrsf1afl/fl and Sharpincpdm/cpdm;Tnfrsf1afl/wt mice that were littermates of the Sharpincpdm/cpdm;TNFR1E-KO mice. The Sharpincpdm/cpdm;Tnfrsf1a−/− mice were derived from a different line and shown here is the picture and the staining from the age-matched mice. Bars represent mean values ± SEM. Statistical significance was determined using the Student's t test (***p ≤ 0.001).DOI:http://dx.doi.org/10.7554/eLife.03422.003
Mentions: Previous studies showed that TNF is required for the development of multi-organ inflammation in Sharpincpdm/cpdm mice (Gerlach et al., 2011). To address whether this function of TNF is mediated by TNFR1, we crossed Sharpincpdm/cpdm mice with Tnfrsf1a−/− animals. Double deficient Sharpincpdm/cpdm;Tnfrsf1a−/− mice did not develop skin inflammation, demonstrating that TNF-induced TNFR1 signaling is essential for the pathogenesis of inflammatory skin lesions in Sharpincpdm/cpdm mice (Figure 1). We then tried to identify the cellular target of the pathogenic TNFR1 signaling in Sharpincpdm/cpdm mice. We have recently shown that TNFR1 signaling in NF-κB-deficient epidermal keratinocytes drives psoriasis-like skin inflammation in mice (Kumari et al., 2013), identifying keratinocytes as an important cellular target of pathogenic TNF signaling in skin inflammation. To address whether TNFR1 signaling in epidermal keratinocytes drives the skin inflammation in Sharpincpdm/cpdm mice, we crossed Sharpincpdm/cpdm mice with K14Cre-Tnfrsf1afl/fl (TNFR1E-KO) mice that lack TNFR1 specifically in keratinocytes (Figure 1A). These Sharpincpdm/cpdm;TNFR1E-KO mice did not develop any macroscopic signs of skin inflammation (Figure 1B). In addition, histological analysis of Sharpincpdm/cpdm;TNFR1E-KO mice skin revealed a normal epidermis without keratinocyte death (cleaved caspase-3 staining in Figure 1C), skin inflammation (F4/80 staining in Figure 1C), or epidermal hyperplasia (H&E, Keratin 6, Keratin 10, and Loricrin staining in Figure 1C and quantification in Figure 1D), similar to Sharpincpdm/cpdm;Tnfrsf1a−/− animals (Figure 1C,D). These results demonstrate that TNFR1 signaling in epidermal keratinocytes is essential for the pathogenesis of skin inflammation in Sharpincpdm/cpdm mice.10.7554/eLife.03422.003Figure 1.Tumor necrosis factor receptor 1 (TNFR1) signaling in keratinocytes triggers chronic proliferative dermatitis phenotype in Sharpincpdm/cpdm mice.

Bottom Line: Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation.Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role.Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, Center for Molecular Medicine, University of Cologne, Cologne, Germany.

ABSTRACT
Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.

Show MeSH
Related in: MedlinePlus